POTASSIUM NUTRITION IN THE OIL PALM: A MOLECULAR PERSPECTIVE
POTASSIUM NUTRITION IN THE OIL PALM:
A MOLECULAR PERSPECTIVE
MOHD NAQIUDDIN*; NYUK LING MA**and MEILINA ONG-ABDULLAH*
ABSTRACT
+
+
+ uptake are the K+ T K+
ptake P igh A K+ (Elaeis guineensis),
Keywords: potassium, KT/KUP/HAK, oil palm, molecular characterisation.
Date received: 23 January 2019; Sent for revision: 24 January 2019; Accepted: 2 October 2019.
* Malaysian Palm Oil Board, 6 Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia.
E-mail: [email protected]
** School of Fundamental Science, Universiti Malaysia Terengganu,
21030 Kuala Terengganu, Terengganu, Malaysia.
INTRODUCTION
Elaeis guineensis or the oil palm is by far the most et al., 2019;
Bourgis et al., 2011; Yan, 2017), produces almost 70 million tonnes of palm oil in 2017 or 30% out of the total world edible oil production, claiming the top spot amongst 16 other vegetable oils as the most oils and fats has seen a staggering increase of 167%
in less than three decades from 83.5 million tonnes in 1991-92 to 223 million tonnes in 2017 (Mielke, 2017) and is expected to continue rising proportionally with the growing world population. Malaysia is the second largest producer of palm oil after Indonesia, and both countries currently account for 85% of the total palm oil production in the world (Yan, 2017). In order to meet the growing demand for oils and fats, expansion of the current 5.8 million hectares of plantation area in Malaysia is not a viable option, thus optimising the existing land to
produce maximum yield is the only alternative for sustainable palm oil production. One of the key areas to improve productivity is mineral nutrition.
+) is one of the most important mineral nutrients which play diverse roles in plants opening and closing of stomata in the leaf (to allow water vapour and waste gases to escape and at the same time to provide plants with the ability to withstand drought conditions), maintaining the turgor pressure of the cell (meaning it keeps plants (Maathuis et al., 1996; Schachtman and Schroeder,
+
is a crucial element in fertilisation, which directly affects bunch weight and bunch number (Lamade et al., 2014). For this reason, palms exposed to
+ deficiency have been reported to produce significantly less oil (Diomande et al + is also involved in the transport of sugar assimilates or
‘food’ from photosynthetic tissues to other parts of the plant (Lamade et al., 2014) such as inflorescence
+ deficiency has been associated with the occurrence of vascular wilt disease, cercospora leaf spot, Ganoderma basal stem rot, and the physiological disorders which cause bunch and plant failure (Rankine and Fairhust, 1999).
Journal of Oil Palm Research Vol. 32 (1) March 2020 p. 139-144 DOI: https://doi.org/10.21894/jopr.2019.0029
+ often is very low (Maathuis, 2009), and to compensate the
+ and other nutrients in soil, large amount of fertilisers are applied. Approximately 0.52 t ha-1 yr-1
an associated cost of approximately USD 185 t-1. total production cost especially in time of weakened Malaysian ringgit (MYR). Besides that, with the growing concern of the deteriorating environment, there has been a drive towards mitigating the use of chemical fertilisers. Hence, palms with enhanced traits are the future planting materials for a more sustainable oil palm industry.
Development of crop less dependent on fertilisers and optimisation of fertilisation has been impeded by current lack of knowledge in the molecular aspects of agriculturally and economically important plants.
Nevertheless, advances in molecular genetics and genomics in recent years such as the publication of the Elaeis guineensis genome in 2013 has provided us with endless opportunities for genomic-driven crop improvement. Moving towards this goal we looked at a few gene candidates responsible for
+ uptake, specifically from the superfamily of + ransporters/ + Uptake Permease/High Affinity
+
transporters are found in bacteria, fungi and plants.
In plants, the transporters can be grouped into four distinct clusters (I-IV). Members of cluster I
+acquisition particularly when
+
Navarro, 2006) and are likely to function as H+ + of cluster II are involved in diverse physiological
+-
+-transport, response to while transporters in cluster III and IV are still not well characterised.
by obtaining: (i) full-length sequences of
using a combination of homology screening from several plant databases and Rapid Amplification of cDNA Ends (RACE), (ii) phylogenetic analyses of the obtained sequences, (iii) genes expression analysis, and (iv) functional characterisation in the heterologous system, Escherichia coli.
MATERIALS AND METHODS
Plant Growth, Total Ribonucleic Acid (RNA) Extraction and cDNA Synthesis
12 hr light (2000 lux) and 12 hr dark cycles, and 60%
humidity prior to the experiment. Approximately 100 mg of root tissues were disrupted using a tissue lyser. RNA was immediately extracted from the disrupted tissues using the RNeasy extraction kit (Qiagen) according to the manufacturer’s templates for cDNA synthesis using the Omniscript manufacturer’s protocol.
Construction of Full-length cDNA of the Candidate Genes
family of potassium transporters from other plant species such as Oryza sativa, Arabidopsis thaliana and were used as a reference to generate gene specific oligonucleotide primers for
manufacturer’s protocol.
Phylogenetic Analysis of EgKUP
against Arabidopsis thaliana databases (www.
arabidopsis.org), identified and named based on the closest homologues from A. thaliana. Sequences different species were downloaded from their respective database websites including rice (http://
crep.ncpgr.cn/crep-cgi/home.pl http://
), tomato (http://chibba.agtec.uga.edu/duplication/), wheat and barley (from NCBI database). Alignment of all the sequences was conducted using Clustal Omega
( ),
and the subsequent phylogenetic tree was built using MEGA5 software, and the Neighbour-Joining et al., 2011). Bootstrap testing was performed with 1000 resampling.
Gene Expression Analysis
media containing three different concentrations of
3 3 for periods
to 5.7 using Ca(OH)2 +
concentration, roots tissues were harvested at 7, 14 and 21 days of growth from experimental and control media. For each test condition 100 mg of ground root/leaf tissues were used for RNA extraction.
then performed, followed by quantitative real-
+ transporters from E. guineensis.
several plant databases such as rice, Arabidopsis, date palm and oil palm itself, total RNA extraction, RACE and PCR techniques had successfully led to the identification of three full-length genes from the and
subjected to phylogenetic analysis together with their counterparts from other species for conformity.
( ).
Regulation of EgKUP at Gene and Protein Levels , we conducted
+-starvation of the oil palm plantlets. Level of expression for
+
expression was markedly increased by seven- and five-fold respectively at 14- and 21-day of treatment (
whether some of the cluster II transporters are also whether the changes of observed at the gene level is translated at the protein level, we conducted functional complementation assay by cloning the full-length of into Escherichia coli
+
cells harbouring were able to complement the cells’ defect at 50 mM and 5 mM but not at the
+-concentration, 1 mM, when tested at pH 7.5. At low pH (pH 5.5), only positive control cells were able to survive. Based on previous studies, high-affinity transporters have certain distinct were normalised against the signal obtained for
GAPDH (oligonucleotide primer sequences) are given in Table 1 and the expression levels were then compared to the control group using the method described by Livak and Schmitten (2001).
Growth Test Assay
, and
were individually cloned into plasmid pBAD24 (oligonucleotide primer sequences) are given in Table 1 and then transformed into the Escherichia coli
+ uptake systems. For bacterial growth tests, at
+, serial dilution drops of strains grown in
+ were inoculated on a solid medium containing 5 mM PO4H3, 0.4 mM MgSO4 4, 1 mM citric acid, 1 mg litre-1 thiamine, 0.2% glycerol, 8 mM asparagine, 20
2Cl, and oligo-elements brought to pH 5.5 or pH 7.5 with HCl or arginine respectively and supplemented with 13 mM arabinose and either 50-,
+
on that described by Senn et al
assay was performed to evaluate whether the defect of E. coli
the introduction of a plasmid harbouring genes.
RESULTS AND DISCUSSION
Identification of the Oil Palm KT/KUP/HAK Transporters
+
uptake and homeostasis, it is essential to obtain the
TABLE 1. OLIGONUCLEOTIDE PRIMER SEQUENCE for (A) QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (qRT-PCR) EXPERIMENT AND (B) CONSTRUCTION OF pBAD24 PLASMIDS CONTAINING EgKUP
Primer identification Sense primer (5’-3’) Antisense primer (5’-3’)
GAATTC CCATGG
CCATGG AAGCTT
TCTAGA AAGCTT
characteristics including (i) upregulation of gene
+ +), and
+ deficient cells under
+ (1 mM and lower) and low pH (pH 5.5).
criteria may indicate that this transporter is not energised by inwardly directed H+ gradients or in other words it may not function as high-affinity H+ + co-transporter, at least when recombinantly expressed in E. coli. A very similar expression profile was reported for another cluster II transporter,
+
but failed to mediate high-affinity transport when expressed heterologously in E. coli et al., 1998).
conclusive before further investigation takes place.
+ concentration and low pH dependent behaviour has been demonstrated for
A. thaliana. In planta
mediates high-affinity transport, however only after the introduction of a point mutation, F130S, that the transporter was able to function as high-affinity
+) in E. coli (Alemán et al., 2014). Nevertheless, the results from expression and functional complementation assays may indicate the
On the other hand, the expression of
and was not affected by changes in
+ conditions. At protein level,
+ transport
+ concentration
+ transport.
CONCLUSION
membrane transporter candidates from the successfully been identified and cloned. All of the
+ transport function based on the functional complementation assay.
reported here fulfill the criteria of being a high- affinity transporter. However, the observation that
is transcriptionally activated in plants at
+ +
+-sufficient environment, because such activation may compensate for low efficiency of
Zea mays Phoenix dactylifera lycopersicon esculentum Hordeum vulgare
Cluster I
Cluster IV
EgKUP8*
PaKUP8 AIKUP8 OsHAK8
ZinHAK8 AiKUP3/KT4 OsHAK3
*EgKUP3 PaKUP3 OsHAK2
OsHAK7 AtHAK5
HvHAK1 ZmHAK1
OsHAK1
PoHAK1 LeHAK5
OsHAK4
OsHAK17
OsHAK6 OsHAK26 ZmHAK4
EgKUP1 1 PoKUP1 1 OsHAK1
1
ZmHAK11 AIKUP1 AIKUP101
Cluster III
Cluster II
growing knowledge in this area will greatly assist us in designing planting materials with improved nutrient efficiency.
ACKNOWLEDGEMENT
to the Director-General of MPOB for permission Bernadette Byrne of the Imperial College, London,
REFERENCES
Alemán, F; Caballero, F; Ródenas, R; Rivero, R M;
1.5 1.0 0.5 0
1.5 1.0 0.5 0
1.5 1.0 0.5 0
and Cs(+) selectivity and confers Na(+) and Cs(+) tolerance to yeast under heterologous expression.
: 430-430. DOI: 10.3389/
fpls.2014.00430.
Banuelos, M A (2002). Inventory and functional
of rice. : 784-795.
F; Drira, N; Ohlrogge, J B and Arondel, V (2011).
Comparative transcriptome and metabolite analysis of oil palm and date palm mesocarp that differ dramatically in carbon partitioning.
: 12527-12532.
Soro, P A and Ebby, N (2004). Application of laser induced fluorescence technique to the study of potassium deficiency of the palm oil tree [Elaeis
guineensis (Jacq)]. : 95-106.
+ +
K+ + +
+ + concentrations; 50 mM, 5 mM and also containing empty vector.
b pH 7.5 pH 5.5
50 mM
7d 14d 21d 7d 14d 21d 7d 14d 21d
Time course Time course Time course
**
**
Control Excess-K+ Depleted-K+
Relative expression
EgKUP3 EgKUP8 EgKUP11
50 mM
EgKUP3 EgKUP8 EgKUP11 +ve control -ve control EgKUP3 EgKUP8 EgKUP11 +ve control -ve control
5 mM 5 mM
1 mM 1 mM
Outlook with Special Emphasis on Palm Oil. 44 pp.
phosphorus, potassium and magnesium in mature
oil palm. : 10-15.
potassium and sodium transport systems in plants.
: 1149-1160.
in fungi and plants. Biochimica et Biophysica Acta : 1-30.
Schachtman, D P and Schroeder, J I (1994). Structure and transport mechanism of a high-affinity potassium uptake transporter from higher plants.
: 655-658.
Navarro, A (2001). Comparative functional features
transporters. : 44563-44569.
DOI: 10.1074/jbc.M108129200.
evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and : 2731-2739. DOI: 10.1093/
molbev/msr121.
Very, A A and Sentenac, H (2003). Molecular higher plants. Annu. Rev. Plant Biol., 54: 575-603.
hated crop. : 306-308.
transporters: Single family - multiple functions.
: 1035-1041.
high-affinity potassium transport activity. Plant Cell, : 51-62.
Nambiappan; Elina Hishamuddin; Mohd Noor Subramaniam; Shamala Sundram and Ghulam performance in Malaysia and R&D progress in 2018.
: 165-194.
Gérardeaux, E (2014). Occurrence of potassium location in oil palm tissues with reserve sugars:
Paper presented at the International Oil Palm Conference, Bali, Indonesia. Paper without proceedings retrieved from http://agritrop.cirad.
fr/574656/
of relative gene expression data using real-time method. : 402-408. DOI: 10.1006/
meth.2001.1262.
Maathuis, F; Verlin, D; Smith, F A; Sanders,
transport. : 1609-1616.
Maathuis, F J M (2009). Physiological functions of mineral macronutrients. Current Opinion in Plant
: 250-258.
